7. Project Summary/Abstract Staphylococcus aureus is the leading cause of life threatening skin and soft tissue infections, osteomyelitis, pneumonia and endocarditis, but spends much of its time as a commensal bacterium, colonizing 30-50% of the population asymptomatically. Vaccine candidates show promise for highly susceptible populations, but a general, effective approach to preventing staphylococcal infections remains elusive. Moreover, the mechanistic basis for the commensal-to-pathogen switch is complex and remains unsolved, but changing nutrient availability in the various environments of the human host likely plays an important role in informing the bacterium's decision to switch between lifestyles. The global transcriptional regulator CodY is activated by isoleucine, leucine, valine (ILV) and GTP, and adjusts metabolism and virulence gene expression. Our preliminary data indicate that CodY activity is not binary (i.e., on or off). Rather, CodY can sense a range of concentrations of ILV and GTP to generate a hierarchical transcriptional and physiological response. That is, under conditions of increasing ILV and GTP depletion, toxins and spreading factors are sequentially induced. Additional work by our lab has shown that CodY controls nearly all of the known virulence genes in conjunction with three of the most important regulators of S. aureus virulence: the Sae two-component system, the regulatory protein Rot, and the Agr quorum-sensing system. In collaboration with staphylococcal biologists Christopher Montgomery (Nationwide Children's Hospital), Victor Torres (NYU School of Medicine), and Taeok Bae (Indiana School of Medicine-NW), we will unravel the mechanistic basis by which CodY ties virulence to nutrient availability. We will use LC-MS based metabolomics, confocal microscopy, classical molecular genetics and biochemistry to (i) determine the mechanisms by which CodY regulates the expression of the sae locus, and (ii) determine how nutrient availability alters the activity of the Sae Two Component System. Additionally, we will explore mechanistically how CodY constrains virulence during S. aureus-immune cell encounters as well as during skin and soft tissue infection using a mouse model of S. aureus dermonecrosis. Understanding how nutrient signals are integrated into the virulence regulatory network by factors like CodY can potentially inform the rational design of novel antimicrobials that limit host damage, desperately replacing obsolete antibiotics in an era of pan-resistance.